Method of protein stabilization

ABSTRACT

The present invention relates to a method of improving heat-stability of a protein by substituting aspartic acid, a hydrophilic amino acid present on the surface of a water-soluble protein, with glutamic acid by the site-specific mutagenesis. The substitution of aspartic acid, a hydrophilic amino acid present on most proteins, with glutamic acid, which is very similar to aspartic acid in both electrostatic feature and molecular weight, can improve the heat stability of a resulting protein without affecting its structure or the function thus enabling to extend its applications to a variety of industrial fields such as medicine, food and chemistry.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method of heat-stabilization of a protein by substituting hydrophilic amino acid residues of a water-soluble protein present on the surface of the protein with another amino acid by site-specific mutagenesis.

[0003] 2. Description of the Related Art

[0004] Proteins are polymers consisting of hydrophilic and hydrophobic amino acid residues and have been widely applied to food, medicine and other biochemical industries due to the varieties of their chemical and biological functions. In industrial applications, however, the stability of a protein has been of much importance because instability of proteins during distribution or use would cause them to be easily inactivated thus incurring various unwanted problems. As a result, enormous efforts have been made to improve the stability of proteins by means of adding additives or inducing mutations. For example, mutations increasing hydrophobic interactions of a protein [Kellis et al., Nature, 333, 784-786, (1988); Karpusas et al., PNAS, 86, 8237-82411], decreasing the unfolded state entropy by introducing a disulfide bond [Matsumara et al., Nature, 342, 291-293, (1989)] or prolins in a protein [Matthews et al., PNAS, 84, 6663-6667, (1987)], or improving the helix dipole and electrostatic interactions [Nicholson et al., Nature, 336, 651-656, (1988)] have been used to improve the stability of a protein. Nevertheless, these methods can affect the structure of a given protein, and simple introductions of these methods without prior understanding of the possible effects on the protein structure may result in protein instability. Therefore, it has been in high demand to find a method to stabilize a protein without affecting the structure or the function of a given protein.

SUMMARY OF THE INVENTION

[0005] The present invention relates to a method of heat-stabilization of a protein, and more particularly, to a method of improving heat-stability of a protein by substituting aspartic acid, a hydrophilic amino acid present on the surface of water-soluble proteins, with glutamic acid by site-specific mutagenesis. The substitution of aspartic acid, a hydrophilic amino acid present on most proteins, with glutamic acid, which is very similar to aspartic acid in both electrostatic feature and molecular weight, can improve the heat stability of a resulting protein without affecting its structure or the function thus enabling to extend its applications to a variety of industrial fields such as medicine, food and chemistry.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006]FIG. 1 shows the procedure of constructing wild type protein expression vectors of M_(j)TRX and ETRX.

[0007]FIG. 2 shows a method of constructing mutant protein expression vectors of M_(j)TRX and ETRX by site-specific mutagenesis by using PCR (polymerase chain reaction).

[0008]FIG. 3 is a graph that shows the heat-stabilities of both a wild type M_(j)TRX and a mutant M_(j)TRX, wherein three glutamic acids (Glu25, Glu36, Glu5l) were substituted with three aspartic acids. Solid line and dotted line show the excess heat capacity of a wild type M_(j)TRX and a mutant M_(j)TRX, respectively.

[0009]FIG. 4 is a graph that shows the heat-stabilities of both a wild type ETRX and a mutant ETRX, wherein three aspartic acids (Asp15, Asp43, Asp47) were substituted with three glutamic acids. Solid line and dotted line show the excess heat capacity of a wild type ETRX and a mutant ETRX, respectively.

[0010]FIG. 5 is a graph that compares the activity between a wild type (λ) M_(j)TRX and a mutant M_(j)TRX (μ).

[0011]FIG. 6 is a graph that compares the activity between a wild type (λ) ETRX and a mutant ETRX (μ).

DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a method of heat-stabilization of a protein, and more particularly, to a method of improving heat-stability of a protein by substituting aspartic acid, a hydrophilic amino acid present on the surface of a water-soluble protein, with glutamic acid by site-specific mutagenesis. Both aspartic acid and glutamic acid are hydrophilic amino acids and thus they are usually present on the surface of most proteins and their characteristics are also very similar to each other. For example, they are both acidic amino acids having similar electrostatic properties and the only difference lies in that glutamic acid is slightly greater in molecular weight than that of aspartic acid by having one more methylene group (−CH₂). Therefore, substituting these hydrophilic amino acid residues by means of a site-specific mutagenesis can improve the heat stability of a resulting protein without affecting its structure or the function even when the precise structure of a given protein is not known.

[0013] This invention is explained in more detail based on the following Examples but they should not be construed as limiting the scope of this invention.

EXAMPLE 1

[0014] Preparation of Methanococcus jannaschii Thioredoxin (M_(j)TRX) and Escherichia coli Thioredoxin (ETRX)

[0015] (1) Preparation of Wild Type Protein Expression Vector

[0016] M_(j)TRX and ETRX genes (SEQ ID NO: 15) were obtained by using genomic DNAs of Methanococcus jannaschii and Escilerichia coli as a DNA template, respectively, under the PCR condition of denaturing at 96° C., annealing at 58° C., and extension at 72° C. Thus obtained thioredoxin genes were then inserted into a NdeI-BamHI restriction site of pET15b (Novagen, USA) to construct an expression vector for the preparation of wild type proteins of Methanococcus jannaschii thioredoxin (M_(j)TRX) and Escherichia coli thioredoxin (ETRX) (see FIG. 1).

[0017] (2) Preparation of M_(j)TR Mutant Protein

[0018] M_(j)TRX mutant protein expression vector was prepared by substituting each or all glutamic acids at position 25, 36, and 51 of the wild type M_(j)TRX protein with aspartic acids by using the expression vector (Novagen, USA) prepared in the above step (1) with inserted M_(j)TRX genes in order to prepare M_(j)TRX mutant protein. Here, DNA PCR technique was employed in substituting GAA, a codon that encodes glutamic acid, with GAT, a codon that encodes aspartic acid, and the oligomers used as DNA primers are as follows (the underlined parts represent substituted codons). [SEQ ID NO.1] E25D-1: 5′-CTAAAAGAGTTGTT GAT GAGGTAGCAAATG-3′ [SEQ ID NO.2] E25D-2: 5′-CATTTGCTACCTC ATC AACAACTCTTTTTAG-3′ [SEQ ID NO.3] E36D-1: 5′-CCGGATGCTGTT GAT GTAGAATACATAAAC-3′ [SEQ ID NO.4] E36D-2: 5′-GTTTATGTATTCTAC ATC AACAGCATCCGG-3′ [SEQ ID NO.5] E51D-1: 5′-CAAAAGGCAATG GAT TATGGGATAATGG-3′ [SEQ ID NO.6] E51D-2: 5′-CCATTATCCCATA ATC CATTGCCTTTTG-3′ [SEQ ID NO.7] T7 Promoter: 5′-TAATACGACTCACTATAGGG-3′ [SEQ ID NO.8] T7 Terminator Code: 5′-GCTAGTTATTGCTCAGCGG-3′

[0019] The reaction solution contains 0.1 μM of dNTP mixture, about 20 ng of pET15b wild type M_(j)TRX plasmid, 1.5 unit of pfu-DNA polymrase (Stratagene, USA) and a buffer solution to make the total reaction volume to 50 μL.

[0020] First, PCR was performed for a reaction solution containing T7 promoter oligomers and oligomers from even-numbered SEQ ID NOs, as well as for another reaction solution containing T7 terminator oligomers and oligomers from odd-numbered SEQ ID NOs under the PCR condition of 60 sec of denaturing at 96° C., 30 sec of annealing at 60° C., 50 sec of extension at 72° C. which were repeated for 30 cycles. From these PCR reactions, amplified 5′- and 3′-DNA fragments with substituted aspartic acid were obtained. Amplified DNA fragments were separated and then placed for the second round of PCR reaction. Here, only the amplified DNA fragments were added into the above reaction solution (without 20 ng of pET15b wild type M_(j)TRX plasmid) with odd-numbered and even-numbered SEQ ID oligomers and PCR was performed for 10 cycles. Then, the third PCR was performed for 30 cycles under the same condition after adding T7 promoter and T7 terminator in the resulting reaction mixture of the second PCR. The mutant M_(j)TRX fragments obtained from the third PCR reaction were inserted into a NdeI-BamHI restriction site of pET15b (Novagen, USA) to prepare the desired protein expression vector (see FIG. 2).

[0021] (3) Preparation of ETRX Mutant Protein

[0022] ETRX mutant protein expression vector was prepared to produce ETRX mutant protein by substituting aspartic acids at position 15, 43, and 47, with glutamic acids by using the expression vector having ETRX gene. The DNA oligomers used are shown below and the method of preparation is the same as in the preparation of MJTRX mutant proteins. [SEQ 9] D15E-1: 5′-CAGTTTTGACACG GAA GTACTCAAAGCGG-3′ [SEQ 10] D15E-2: 5′-CCGCTTTGAGTAC TTC CGTGTCAAAACTG-3′ [SEQ 11] D43E-1: 5′-CGCCCCGATTCTG GAA GAAATCGCTGACG-3′ [SEQ 12] D43E-2: 5′-CGTCAGCGATTTC TTC CAGAATCGGGGCG-3′ [SEQ 13] D47E-1: 5′-CTGGATGAAATCGCT GAA GAATATCAGGGCAAAC-3′ [SEQ 14] D47E-2: 5′-GTTTGCCCTGATATTC TTC AGCGATTTCATCCAG-3′

[0023] All the mutant DNA sequences were confirmed by using an auto sequencer ABI373-DNA (Perkin Elmer, USA).

EXAMPLE 2

[0024] Production and Separation of a Protein

[0025] The expression vectors obtained in the Example 1 were transformed into E. coli BL21(DE3). The transformed E. coli BL21(DE3) were cultured at 37° C. in LB broth containing 100 μg/mL of ampicillin until they reached an exponential growth phase (OD₆₀₀≡1.0), and then added with 0.4 mM isopropyl β-D-thiogalactopyranoside (IPTG) for the expression of wild type and mutant proteins of M_(j)TRX and ETRX. Thus obtained proteins were then separated by using a nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity column (Qiagen, USA) and a Superdex G75 (Pharmacia, USA) gel filtration column, and both wild type and mutant MjTRX and ETRX proteins having at least 95% purity were obtained about 10 mg per liter culture broth [Lee et al. (2000), Biochemistry, 39, 6652-6659].

Experimental Example 1

[0026] Comparison of Thermodynamic Stability of Proteins

[0027] About 1 mg/mL of proteins obtained in the above Example 2 were placed in 50 mM potassium phosphate buffer solution of pH 6.5, and the excess heat capacity was obtained using a differential scanning calorimetry with the heating rate of 1° C./min. The results showed that the transition temperature (T_(m)) of the wild type M_(j)TRX was 116° C. while that of the mutant M_(j)TRX wherein three glutamic acids were all replaced with aspartic acids was 109° C. This is shown in FIG. 3, wherein the solid line represents a wild type M_(j)TRX while the dotted line indicates a mutant M_(j)TRX, and the T_(m) of the mutant M_(j)TRX decreased by 2° C. per each substitution of glutamic acid with aspartic acid (Table 1). TABLE 1 MjTRX Protein wild type E25D E25/36D E25/36/51D Transition 116.1 ± 0.3 114.4 ± 0.4 111.9 ± 0.3 109.4 ± 0.5 Temperature

[0028] In FIG. 4, the solid line represents a wild type ETRX while the dotted line indicates a mutant ETRX, and the T_(m) of the mutant ETRX increased by 2° C. per each substitution of aspartic acid with glutamic acid; i.e., the T_(m) of the mutant ETRX, wherein three aspartic acids were replaced by glutamic acids, was raised to 92° C. from the 86° C. of the wild type ETRX, thus showing a total increase of 6° C. in T_(m) (Table 2). TABLE 2 ETRX Protein wild type D15E D43/47E D15/43/47E Transition 86.4 ± 0.2 87.5 ± 0.1 90.4 ± 0.1 91.8 ± 0.2 Temperature

Experimental Example 2

[0029] The Comparison of Protein Activities

[0030] Insulin-reduction activity assay was used to measure the activities of M_(j)TRX and ETRX for both a wild type and a mutant, which were produced in the Example 2. The protein concentration used in this assay was 2 μM. The degree of insulin reduction with time was measured at 650 nm and the results are shown in FIGS. 5 and 6. FIG. 5 shows the results of the assay for the activities of M_(j)TRX while FIG. 6 shows that of ETRX. From the FIG. 5 it is shown that there is little difference in protein activity between a wild type and a mutant for M_(j)TRX (FIG. 5) while there is a slight difference of about 7% noticed for ETRX, which in fact does not appear to be of much significance.

[0031] As described above, the present invention relates to a method of improving heat-stability of water-soluble proteins. According to the present invention, substitution of aspartic acid, a hydrophilic amino acid present on most proteins, with glutamic acid, which is very similar to aspartic acid in both electrostatic feature and molecular weight, can improve the heat stability of a resulting protein, even when its precise structure is not known, without affecting its structure or the function thus enabling to extend its applications to a variety of industrial fields such as medicine, food and chemistry.

1 16 1 30 DNA Artificial Sequence Description of Artificial Sequence Primer 1 ctaaaagagt tgttgatgag gtagcaaatg 30 2 31 DNA Artificial Sequence Description of Artificial Sequence Primer 2 catttgctac ctcatcaaca actcttttta g 31 3 30 DNA Artificial Sequence Description of Artificial Sequence Primer 3 ccggatgctg ttgatgtaga atacataaac 30 4 30 DNA Artificial Sequence Description of Artificial Sequence Primer 4 gtttatgtat tctacatcaa cagcatccgg 30 5 28 DNA Artificial Sequence Description of Artificial Sequence Primer 5 caaaaggcaa tggattatgg gataatgg 28 6 28 DNA Artificial Sequence Description of Artificial Sequence Primer 6 ccattatccc ataatccatt gccttttg 28 7 20 DNA Artificial Sequence Description of Artificial Sequence Primer 7 taatacgact cactataggg 20 8 19 DNA Artificial Sequence Description of Artificial Sequence Primer 8 gctagttatt gctcagcgg 19 9 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 9 cagttttgac acggaagtac tcaaagcgg 29 10 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 10 ccgctttgag tacttccgtg tcaaaactg 29 11 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 11 cgccccgatt ctggaagaaa tcgctgacg 29 12 29 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 12 cgtcagcgat ttcttccaga atcggggcg 29 13 34 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 13 ctggatgaaa tcgctgaaga atatcagggc aaac 34 14 34 DNA Artificial Sequence Description of Artificial Sequence Synthetic oligonucleotide 14 gtttgccctg atattcttca gcgatttcat ccag 34 15 327 DNA Escherichia coli CDS (1)..(324) 15 agc gat aaa att att cac ctg act gac gac agt ttt gac acg gat gta 48 Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val 1 5 10 15 ctc aaa gcg gac ggg gcg atc ctc gtc gat ttc tgg gca gag tgg tgc 96 Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys 20 25 30 ggt ccg tgc aaa atg atc gcc ccg att ctg gat gaa atc gct gac gaa 144 Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu 35 40 45 tat cag ggc aaa ctg acc gtt gca aaa ctg aac atc gat caa aac cct 192 Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro 50 55 60 ggc act gcg ccg aaa tat ggc atc cgt ggt atc ccg act ctg ctg ctg 240 Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu 65 70 75 80 ttc aaa aac ggt gaa gtg gcg gca acc aaa gtg ggt gca ctg tct aaa 288 Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys 85 90 95 ggt cag ttg aaa gag ttc ctc gac gct aac ctg gcg taa 327 Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105 16 108 PRT Escherichia coli 16 Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp Val 1 5 10 15 Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp Cys 20 25 30 Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp Glu 35 40 45 Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn Pro 50 55 60 Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu Leu 65 70 75 80 Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser Lys 85 90 95 Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala 100 105 

What is claimed is:
 1. A method of heat-stabilization of a protein wherein said method comprises a substitution of aspartic acid, a hydrophilic amino acid present on the surface of a water-soluble protein, with glutamic acid. 